Our long range goal is to understand the molecular mechanisms by which actin and its associated proteins participate in cellular movements and the structure of cytoplasm. We will use Acanthamoeba as our model system. To clarify the mechanism of actin polymerization, we will characterize nucleotide hydrolysis during polymerization, reconcile the mechanism of elongation that we deduced from measurements in the electron microscope with observations on bulk samples, analyze the mechanism of nucleation by direct methods, characterize the dynamics of actin filaments at seady state and study the mechanisms of action of cytochalasin and phalloidin. To understand the functions of the actin monomer binding protein called profilin, we will determine the 3-D structure of Acanthamoeba profilinI, profilin-II and ventebrate profilin at atomic resolution by X-ray crystallography, identify amino acid contacts between profilin and actin by chemical crosslinking and protein sequencing so that we can establish how the proteins bind together, characterize the dynamics of the binding of profilin to actin monomers and filaments and search for factors that might regulated this interaction, localize the 2 profilin isoforms in Acanthamoeba and characterize a library of monoclonal antibodies to profilins with the goal of identifying inhibitors of actinprofilin interaction for use in other experiments. Actophorin is an actin monomer binding and filament severing protein. We will determine the primary structure of actophotin from a cloned cDNA, determine the 3-D structure of actophorin at atomic resolution, identify amino acid contacts between actophorin and actin by chemical crosslinking and protein sequencing so that we can show how the proteins bind together, characterize the interaction of actophorin with actin monomers and filaments as a model for the mechanisms of other severning proteins, search for factors that might regulate the interaction of actophorin and actin and localize actophorin in Acanthanoeba. To characterize the actin filament crosslinking protein called alphaactinin, we will study the mechanical properties of mixtures of alphaaclinin and actin filaments as a function of filament length and concentation, analyze the dynamics of the interaction of alphaactinin with actin filaments by fluroescence recovery after photobleaching, stopped flow kinetics, equilibrium binding and electron microscopy, search for factors regulating the interaction of alphaactinin with actin filaments and document the intracellular dynamics of alphaactinin by microinjection of labeled protein and photobleaching recovery.